Connexin proteins form the gap junction channels that mediate direct intercellular molecular communication crucial in development, physiology and response to trauma/inflammation/disease. Mutations in connexins cause human pathologies. Elucidation of the molecular mechanisms that regulate connexin channel function is essential for understanding their roles in human physiology and pathophysiology, and to identify targets for translational and basic science studies. The long-term goal of this project is to understand the cytosolic inter-domain interactions of connexin proteins that control channel gating. In connexin43 (Cx43), interactions between the cytoplasmic loop (CL) and the C-terminal domain (CT) is crucially involved physiological function of the channel and is a target for cardiovascular therapies. However, the roles and mechanisms of CL-CT interactions in modulation of other connexin channels, just as likely to be biomedically important, have not been explored. We have found that Cx26 channels are modulated by CL-CT interactions. Cx26 and Cx43 are representative members of the two largest families of connexins. Though structurally analogous, the effects of CL-CT interaction on Cx26 channel function seem to be fundamentally different from those in Cx43, suggesting that CL-CT interaction is a common modulatory mechanism in connexins, yet operates in connexin-specific ways. We propose to elucidate the molecular mechanisms of CL-CT control of channel function using Cx26 and its closely related isoform Cx32. Cx26 is the only connexin channel for which there is a high-resolution structure, making it the cornerstone for structure-based studies of all connexin channels. The proposed studies explore the basis and mechanisms of CL-CT interactions and channel properties they modulate, using strategies successfully applied to other channels, including macroscopic and single channel recordings, use of competing peptides, engineered Cys linkages and mutational analysis. The experiments utilize intact channels, complemented by peptide NMR. We propose to (a) determine the involvement of CL-CT interactions in channel gating, (b) identify the sites of CL-CT interactions, and (c) determine how CL-CT interaction and its effects are altered by mutations that cause human disease. Cx26 and Cx32 are widely distributed in the body. Cx26 mutations cause over half the inherited non-syndromic sensorineural deafness worldwide, and serious disfiguring skin disorders. Cx32 mutations cause a peripheral demyelination. Significantly, in both connexins many disease-causing mutations are in the CL and CT domains. Both connexins are involved in a wide range of pathological and physiological processes, so understanding the basis for their dysregulation has broad biomedical implications, for these connexins and others. The large number of disease causing mutations and the extensive experience of both PIs in studying gating and permeability of Cx26 and Cx32 channels provide a basis for productive, informative investigation of CL-CT interactions, how they are altered by pathological mutations and the effects on channel function.